Reaction mass of didodecyl 3,3'-thiodipropionate and dioctadecyl 3,3'-thiodipropionate and octadecyl 3-[[3-(dodecyloxy)-3-oxopropyl]thio]propionate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL = 20 mg/kg bw/d (2-generation reproduction toxicity study, oral, rat, NaTG)

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target and source substances have similar toxicological properties because:
- all substances are small organic molecules;
- they share structural similarities with common functional groups: one or more thiol and/or thioether group(s) and carboxylic acid (as free acid, salt or ester);
- the metabolism (i.e. ester hydrolysis) leads to comparable products (sulfur-containing core structure in its acid form and alcohols of differing chains lengths)
- covalent protein binding via S-S bonds may be a common mode of action and or chelation of divalent cations via sulfur

The substances were assigned to subgroups according to their main structural features (see Table 1); further justification for subgrouping based on toxicological properties is given below:
- TGA family: Thioglycolic acid, its salts and esters
- 3-MPA family: 3-Mercaptopropionic acid, its salts and esters
- TLA family: Thiolactic acid and its salts
- Intramolecular-S family: Thiodiglycolic acid or Dithiodiglycolic acid and its esters, Thiodipropionic acid or Dithiodipropionic acid and its esters, Methylene bis(butyl thioglycolate)
- Mercaptanes: Thioglycerol, Bis(2-mercaptoethyl) sulfide, 4-Mercaptomethyl-3,6-dithia-1,8-octanedithiol

The acids and salts will dissociate to the respective Thioglycolate or 3-Mercaptopropionate or Thiolactate and the corresponding cation.
In case of the esters, the metabolism expected to occur is ester hydrolysis resulting in the corresponding acid and alcohol.

It was demonstrated, that PETMP and 3-MPA strongly bind to plasma proteins (e.g. via S-S bond to cysteine) in vitro, which is well known for substances containing free SH-groups (Bruno Bock, 2014). Strong protein binding is also expected to occur with the other substances assessed within this paper. The members of the intramolecular-S family are an exception, as they do not contain free SH-groups – protein binding may be less relevant for this family.

Overall, based on close structural similarities, a read-across from the existing repeated dose and reproduction toxicity studies is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see cross-reference -> supporting information

3. ANALOGUE APPROACH JUSTIFICATION
see cross-reference -> supporting information

4. DATA MATRIX
see cross-reference -> supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

not measured/tested

Dose descriptor:

NOEL

Generation:

F1

Effect level:

20 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

Dose descriptor:

NOEL

Generation:

F2

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Reproductive effects observed:

no

Executive summary:

With the exception of the mercaptan family, data on toxicity to reproduction and/or development are available for all subgroups.

 

No effects on mating, fertility, estrous cycle and sperm parameters were observed in any of the studies. There was no evidence of damage to the reproductive organs. Nor did any of the substances show developmental toxicity or teratogenicity.

 

The effects observed in the reproduction/developmental toxicity screening test and the 2-generation reproduction toxicity test with NaTG consisting of a significantly longer gestation period, a non-significantly lower number of corpora lutea, a significantly lower number of implantations and reduced pup survival were only seen in the presence on severe maternal toxicity including mortality and thus, secondary effects.

In a comparable manner, mean live litter size, postnatal survival and pup body weights and body weight gains were reduced in the high dose groups of the reproduction / developmental toxicity screening test with 2-EHTG in the presence of marked general toxicity (mortality).

 

Also, the increase in fetal variations observed in the dermal prenatal developmental toxicity test with NaTG (slightly increased incidences of extra or rudimentary ribs on Lumbar I and of dumbbell cartilage and bipartite ossification in thoracic centra - common skeletal variations in term rat fetuses), is interpreted as secondary to intrauterine growth retardation resulting from maternal toxicity, especially in combination with the absence of teratogenic alterations and with reductions in maternal and fetal weight.

 

Similar effects were seen for the members of the 3-MPA family. Lower mean male fetal weight, increased incidence of fetuses showing incomplete ossification of the thoracic centrum or less than four ossified caudal vertebrae, increased incidence of absent renal papilla were observed in the prenatal developmental toxicity study with PETMP in the high dose in the presence of marked general toxicity in dams.

 

TLA did not cause effects to the reproductive organs in a 28 d repeated dose toxicity study.

 

E12 caused no developmental toxicity in rat, mouse, hamster, and rabbit. Noeffects tothereproductive organswere noted in a 13 wk repeated dose toxicity study.Di-2-EHDTDG did not cause effects tothereproductive organsin a 28 d repeated dose toxicity study.

 

Overall, it can be stated, that this whole group of substances has a very low potential to cause reproductive or developmental toxicity. Effects (e.g., lower fetal weight, intrauterine growth retardation, reduced postnatal survival) were observed only in the presence of severe general maternal toxicity.

 

Based on the available data, this group of substances does not need to be classified for toxicity to reproduction or developmental toxicity.

 

For better comparison, the NO(A)ELs have been recalculated on the basis of S-content, which is assumed to be the main driver for toxicity.

 

Overall comparison of NO(A)ELs (toxicity to reproduction)

Family	Substance (study)	NO(A)EL [mg/kg bw/d]	S in molecule [%]	Related to S-content [mg/kg bw/d]
TGA	ATG (NOEL, OECD TG 414, oral: gavage, rat)	75	32.1	24.1
	NaTG (NOAEL, OECD TG 416, oral: gavage, rat)	20	30.7	6.1
	GMT (reproduction NOEL, OECD TG 422, oral, rat)	50	21.1	10.5
	2-EHTG (reproduction NOAEL, OECD TG 421, oral: gavage, general toxicity, rat)	50	17.1	8.6
3-MPA	MMP (reproduction NOAEL, OECD TG 422, oral: gavage, rat)	100	29.1	29.2
	PETMP (fertility NOAEL, OECD TG 408, oral: gavage, rat)	200	7.2	14.3
	PETMP (developmental NOAEL, OECD TG 414, oral: gavage, rat)	120	7.2	8.6
TLA	TLA (fertility parameters NOAEL, OECD TG 407, oral:gavage, rat)	250	33.0	82.5
Intra-molecular-S	E12 (developmental NOAEL, OECD TG 414, oral: gavage, rat, mouse, hamster, rabbit)	>/=1600	6.8	108.8
	E12 (fertility parameters NOEL, OECD TG 408, oral:gavage, rat)	1000	6.8	68
	Di-2-EHDTDG (fertility parameters NOAEL, OECD TG 407, oral:gavage, rat)	200	9.3	18.7

 

After adjustment to S-content, the NOAELs of most of the substances were within the same order of magnitude. Using the lowest NOAEL obtained in the 2-generation reproduction toxicity study conducted with NaTG is considered to be an appropriate starting point for DNEL derivation. Remaining DNEL uncertainties due to read-across can be adequately considered by applying an additional assessment factor.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available key studies are reliable or reliable with restrictions (Klimisch 1 – 2) and were conducted according to or similar to guidelines.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

For the assessment of toxicity to reproduction, data are available from NaTG, GMT, 2-EHTG, MMP, PETMP, TLA, E12, and Di-2-EHDTDG. A justification for read-across is attached to IUCLID section 13.

 

In the reproduction/developmental toxicity screening test with NaTG (0, 20, 40, 80 mg/kg bw/d) mortality was observed in the high dose (2 males + 6 females; one further female sacrificed due to dead pups) and mid dose (1 female). There were no treatment related effects estrous cyclicity, time to mating, pregnancy index. Females given 80 mg/kg bw/d had a significantly longer gestation period (22.8, p<0.01,vs.21.6 days), a non-significantly lower number ofcorpora lutea(mean of 6.7, ns,vs.8.5) and a significantly lower number of implantations (10.3, p<0.001,vs.16.5) and pups (9.0, p<0.01,vs.14.7). One female had total resorptions and one litter died on day 1post-partum. There were no treatment-related pup clinical signs or necropsy findings. Pups treated at 40 or 80 mg/kg bw/d had higher mean body weight gains than the controls between day 1 and day 5post-partum. There were no effects of treatment on sperm morphology, motility or counts. The mean absolute seminal vesicle weights were statistically significantly lower for all male groups in a dose-related manner (absolute weights were -17%, -19% and -35% at 20, 40 and 80 mg/kg/day, respectively). This correlated with a slight decrease in secretory content in the seminal vesicles observed at microscopic examination of the males given 80 mg/kg/day. In the absence of atrophy of seminal epithelium at microscopic examination, these minor findings were not considered to be adverse.

Male reproductive performance was not affected by treatment with NaTG. Dosing at 40 and 80 mg/kg bw/d resulted in deaths in late gestation associated with delayed delivery. NOEL(female reproductive performance) = 20 mg/kg bw/d, NOEL(toxic effects on progeny) = 40 mg/kg bw/d

 

In the 2-generation reproduction toxicity test with NaTG (0, 10, 20, 40 mg/kg bw/d) 40 mg/kg bw/d had no effect on non-pregnant, naïve, adult rats but that it causes maternal toxicity and death of susceptible pregnant females around the time of delivery.

Effects on the dam include lack of nesting/nursing behavior and this causes death of

the pups which have been delivered. If treatment is stopped just prior to delivery, the females may survive delivery but pup death may still occur and pup clinical signs of coldness suggest that maternal nesting/nursing behavior is still impaired by treatment with the test item and affect pup survival. There is evidence that female rats are more affected by test item treatment than males as shown by lower F1 female body weight and body weight gain.

Minimal to moderate periportal hepatocellular microvacuolation was observed in females and some male F0 animals treated at 40 mg/kg bw/d suggestive of mild hepatotoxicity. There were no effects on sperm parameters in the control or high-dose group males of either generation. There were no effects of treatment on any parameters measured in either males or females with the test item at 10 or 20 mg/kg bw/d.

The small effects observed on pup survival at 40 mg/kg bw/d may have been secondary to the severe and lethal effects observed in the pregnant dams at that dose-level.

NOEL(parental toxicity, female fertility and gestation, development, growth and survival of the progeny) = 20 mg/kg bw/d

NOEL(male fertility, female mating behavior) >/= 40 mg/kg bw/d

 

In the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test with GMT (0, 15, 50, 150 mg/kg bw/d),5 females at 150 mg/kg bw/d died, the cause for death not being evident from histopathology. No effects were observed on mating, offspring litter size and viability, development, clinical signs of the offspring. The NOEL for reproductive effects in males was 150 mg/kg bw/d as assessed by the absence of adverse effects on fertility or on testicular weight and histopathology. Due to the high maternal mortality and toxicity at 150 mg/kg bw/d in the females, which precluded reproductive assessment at that dose level, the NOEL for reproductive toxicity in females is 50 mg/kg bw/d.

 

In the reproduction/developmental toxicity screening test with 2-EHTG (0, 10, 50, 150 mg/kg bw/d) 3 males and 3 females died in the high dose group. No test item-related effects on male and female mating and fertility indices, male copulation index or female conception index, the mean number of days between pairing and coitus, mean gestation lengths, mean numbers of corpora lutea, implantation sites were observed. Mean live litter size in the high dose was reduced in a test item-related manner. Postnatal survival in this group was reduced on PND 0-1 and birth to PND 4, due in part to 1 female with total litter loss on PND 1. Mean male and female pup body weights and body weight gains in the 150 mg/kg/day group were reduced. Several of these pups were also noted as being small. Mean live litter size and postnatal survival in the low and mid dose were unaffected by test item administration. There were no test article-related effects on the general physical condition of the pups or pup body weights in the 10 and 50 mg/kg/day groups. At the necropsy of pups that were found dead, there were no internal findings that were related to parental treatment with the test item. NOAEL(reproductive, developmental, systemic, neonatal toxicity) = 50 mg/kg bw/d

 

In the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test with MMP (0, 25, 50, 100 mg/kg bw/d) no treatment-related effects on precoital time, fertility indices, mean duration of gestation, number of implantations, postimplantation loss, pup survival or litter size from birth through to scheduled sacrifice on day 4 post partum, mean number of corpora lutea per dam were observed. No abnormal findings were noted for pups at first litter check or during the first 4 days post partum. No effects were noted on sex ratios, mean pup weights on day 0 and day 1 post partum, mean pup weight development during the first 4 days post partum. The NOEL for reproduction/developmental toxicity was considered to be 100 mg/kg bw/d.

 

In the 90 d repeated dose toxicity study with PETMP (0, 12.5, 50, 200 mg/kg bw/d) additionally assessing reproductive parameters, no adverse test item-related effects to the reproductive organs were found for any parameter measured, especially regarding organ weights of ovary and testes and histopathology of gonads, uterus, mammary gland, prostate and seminal vesicle.

 

For TLA, data on fertility parameters from a 28 d repeated dose toxicity study (0, 15, 150 and 500/250 mg/kg bw/d) are available:No effects to the reproductive organs were found for any parameter measured, especially regarding organ weights of ovaries, epididymides and testes and histopathology ofovaries, uterus,epididymides, prostate, seminal vesicles and testes. The NOEL (fertility parameters) is 250 mg/kg bw/d.

 

In the 13 wk repeated dose toxicity study with E12 (0,125, 350, 1000 mg/kg bw/d), no histological changes were noted onepidiymides, mammary glands, ovaries, prostate, seminal vesicles, testes, uterus (horn + cervix) up to the highest administered dose of 1000 mg/kg bw/d.

 

In a 28 d toxicity study + 2 wk recovery period (0, 10, 50, 200, 800 mg/kg bw/d) with Di-2-EHDTDG (Ciba-Geigy, 1992) 2/10 males and 5/10 females died in the 800 mg/kg bw/d dose group. Due to mortality and marked signs of toxicity, the 800 mg/kg bw/d dose group was terminated. Body weight, food and water consumption were reduced in this group. At necropsy fatty change of perilobular region in liver, hyperplasia/ulceration of non-glandular stomach, atrophy of testis and thymus were observed. NOAEL(fertility parameters) = 200 mg/kg bw/d.

 

Summary

With the exception of the mercaptan family, data on toxicity to reproduction and/or development are available for all subgroups.

 

No effects on mating, fertility, estrous cycle and sperm parameters were observed in any of the studies. There was no evidence of damage to the reproductive organs. Nor did any of the substances show developmental toxicity or teratogenicity.

 

The effects observed in the reproduction/developmental toxicity screening test and the 2-generation reproduction toxicity test with NaTG consisting of a significantly longer gestation period, a non-significantly lower number ofcorpora lutea,a significantly lower number of implantations and reduced pup survival were only seen in the presence on severe maternal toxicity including mortality and thus, secondary effects.

In a comparable manner, mean live litter size, postnatal survival and pup body weights and body weight gains were reduced in the high dose groups of thereproduction / developmental toxicity screening test with 2-EHTG in the presence of marked general toxicity (mortality).

 

Also, the increase infetal variations observed in the dermal prenatal developmental toxicity test with NaTG (slightly increased incidences of extra or rudimentary ribs on Lumbar I and of dumbbell cartilage and bipartite ossification in thoracic centra - common skeletal variations in term rat fetuses), is interpreted as secondary to intrauterine growth retardation resulting from maternal toxicity, especially in combination with the absence of teratogenic alterations and with reductions in maternal and fetal weight.

 

Similar effects were seen for the members of the 3-MPA family. Lower mean male fetal weight, increased incidence of fetuses showing incomplete ossification of the thoracic centrum or less than four ossified caudal vertebrae, increased incidence of absent renal papilla were observed in the prenatal developmental toxicity study with PETMP in the high dose in the presence of marked general toxicity in dams.

 

TLA did not cause effects tothereproductive organsin a 28 d repeated dose toxicity study.

 

E12 caused no developmental toxicity in rat, mouse, hamster, and rabbit. Noeffects tothereproductive organswere noted in a 13 wk repeated dose toxicity study.Di-2-EHDTDG did not cause effects tothereproductive organsin a 28 d repeated dose toxicity study.

 

Overall, it can be stated, that this whole group of substances as described inTable1Substance identitieshas a very low potential to cause reproductive or developmental toxicity. Effects (e.g., lower fetal weight,intrauterine growth retardation,reduced postnatal survival) were observed only in the presence of severe general maternal toxicity.

 

Based on the available data, this group of substances does not need to be classified for toxicity to reproduction or developmental toxicity.

 

For better comparison, the NO(A)ELs have been recalculated on the basis of S-content, which is assumed to be the main driver for toxicity.

 

Overall comparison of NO(A)ELs (toxicity to reproduction)

Family	Substance (study)	NO(A)EL [mg/kg bw/d]	S in molecule [%]	Related to S-content [mg/kg bw/d]
TGA	ATG (NOEL, OECD TG 414, oral: gavage, rat)	75	32.1	24.1
	NaTG (NOAEL, OECD TG 416, oral: gavage, rat)	20	30.7	6.1
	GMT (reproduction NOEL, OECD TG 422, oral, rat)	50	21.1	10.5
	2-EHTG (reproduction NOAEL, OECD TG 421, oral: gavage, general toxicity, rat)	50	17.1	8.6
3-MPA	MMP (reproduction NOAEL, OECD TG 422, oral: gavage, rat)	100	29.1	29.2
	PETMP (fertility NOAEL, OECD TG 408, oral: gavage, rat)	200	7.2	14.3
	PETMP (developmental NOAEL, OECD TG 414, oral: gavage, rat)	120	7.2	8.6
TLA	TLA (fertility parameters NOAEL, OECD TG 407, oral:gavage, rat)	250	33.0	82.5
Intra-molecular-Sulfur	E12 (developmental NOAEL, OECD TG 414, oral: gavage, rat, mouse, hamster, rabbit)	>/=1600	6.8	108.8
	E12 (fertility parameters NOEL, OECD TG 408, oral:gavage, rat)	1000	6.8	68
	Di-2-EHDTDG (fertility parameters NOAEL, OECD TG 407, oral:gavage, rat)	200	9.3	18.7

 

After adjustment to S-content, the NOAELs of most of the substances were within the same order of magnitude. Using the lowest NOAEL obtained in the 2-generation reproduction toxicity study conducted with NaTG is considered to be an appropriate starting point for DNEL derivation. Remaining DNEL uncertainties due to read-across can be adequately considered by applying an additional assessment factor.

 

 

References

Arkema, 2010. Two-generation reproduction toxicity study by oral route (gavage) in rats. Unpublished report. CIT 35047 RSR

 

BASF, 1993. 4 week oral (gavage) dose range-finding study in the rat. Unpublished report. Pharmakon Europe 380/572

 

Bibra, 1988. A 28-day study in rats with thioglycolic acid, 2-ethylhexyl ester including investigation of hepatic peroxisomal activity. Unpublished report. Bibra 689/1/88

 

Bruno Bock, 2000. Thiolactic acid 98/99%: Twenty-eight day repeated dose oral (gavage) toxicity study in the rat.Unpublished report. SPL 1158/028.

 

Bruno Bock, 2010. 13-week toxicity study by oral route (gavage) in rats followed by a 4-week treatment-free period. Unpublished report. CIT 34814

 

Bruno Bock, 2010b. Reproduction/developmental toxicity screening test by oral route (gavage) in rats. Unpublished report. CIT 30721 RSR

 

Bruno Bock, 2015b.PETMP: 13-Week Oral (Gavage) Toxicity Study in the Wistar Rat with a 4-Week Recovery Period.Unpublished report. HarlanD89724

 

Ciba-Geigy, 1992. 28 days subactute, oral toxicity study in rats (gavage). Unpublished report. Ciba-Geigy 894135

 

Mitsui Toatsu Chemicals Incorporated, 1991. GST: Four-week toxicity and reversibility study following oral administration to rats. Unpublished report. LSR 90/MT0050/1286

 

NTP, 2003. Thirteen-week subchronic dermal toxicity study of sodium thioglycolate (NaT) in Fisher 344 rats and B6C3F1 mice. Unpublished report. BioReliance 98007.03/99-98007.04

 

Thioesters Association, 2001a. IUCLID data set for 3,3’-thiodipropionic acid, didodecyl ester, submission to US EPA

 

Thioesters Association, 2001b. IUCLID data set for 3,3’-thiodipropionic acid, dioctadecyl ester, submission to US EPA

 

Thioesters Association, 2005. 2-Ethylhexyl mercaptoacetate [CAS No. 7659-86-1]: a reproduction/developmental toxicity screening study in rats. Unpublished report. WIL-528002

 

Thioesters Association, 2007a. Glyceryl thioglycolate 80%: oral (gavage) combined repeat dose toxicity study with reproduction/developmental toxicity screening test in the rat. Unpublished report. SPL 2207/0003

 

Thioesters Association, 2007b.Methyl 3-Mercaptopropionate:Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat. Unpublished report.RCC A74698

 

WHO, 1962. Evaluation of a number of antimicrobials and antioxidants. Sixth report of the Joint FAO/WHO Expert Committee on Food Additives.

Effects on developmental toxicity

Description of key information

NOAEL = 20 mg/kg bw/d (2-generation reproduction toxicity study, oral, rat, NaTG)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target and source substances have similar toxicological properties because:
- all substances are small organic molecules;
- they share structural similarities with common functional groups: one or more thiol and/or thioether group(s) and carboxylic acid (as free acid, salt or ester);
- the metabolism (i.e. ester hydrolysis) leads to comparable products (sulfur-containing core structure in its acid form and alcohols of differing chains lengths)
- covalent protein binding via S-S bonds may be a common mode of action and or chelation of divalent cations via sulfur

The substances were assigned to subgroups according to their main structural features (see Table 1); further justification for subgrouping based on toxicological properties is given below:
- TGA family: Thioglycolic acid, its salts and esters
- 3-MPA family: 3-Mercaptopropionic acid, its salts and esters
- TLA family: Thiolactic acid and its salts
- Intramolecular-S family: Thiodiglycolic acid or Dithiodiglycolic acid and its esters, Thiodipropionic acid or Dithiodipropionic acid and its esters, Methylene bis(butyl thioglycolate)
- Mercaptanes: Thioglycerol, Bis(2-mercaptoethyl) sulfide, 4-Mercaptomethyl-3,6-dithia-1,8-octanedithiol

The acids and salts will dissociate to the respective Thioglycolate or 3-Mercaptopropionate or Thiolactate and the corresponding cation.
In case of the esters, the metabolism expected to occur is ester hydrolysis resulting in the corresponding acid and alcohol.

It was demonstrated, that PETMP and 3-MPA strongly bind to plasma proteins (e.g. via S-S bond to cysteine) in vitro, which is well known for substances containing free SH-groups (Bruno Bock, 2014). Strong protein binding is also expected to occur with the other substances assessed within this paper. The members of the intramolecular-S family are an exception, as they do not contain free SH-groups – protein binding may be less relevant for this family.

Overall, based on close structural similarities, a read-across from the existing repeated dose and reproduction toxicity studies is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see cross-reference -> supporting information

3. ANALOGUE APPROACH JUSTIFICATION
see cross-reference -> supporting information

4. DATA MATRIX
see cross-reference -> supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

histopathology: non-neoplastic

mortality

other: nursing behaviour

Abnormalities:

no effects observed

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in postnatal survival

Abnormalities:

no effects observed

Developmental effects observed:

no

Executive summary:

With the exception of the mercaptan family, data on prenatal developmental toxicity are available for all subgroups.

 

None of the substances showed developmental toxicity or teratogenicity.

 

The effects observed in the reproduction/developmental toxicity screening test and the 2-generation reproduction toxicity test with NaTG consisting of a significantly longer gestation period, a non-significantly lower number of corpora lutea, a significantly lower number of implantations and reduced pup survival were only seen in the presence on severe maternal toxicity including mortality and thus, secondary effects.

In a comparable manner, mean live litter size, postnatal survival and pup body weights and body weight gains were reduced in the high dose groups of the reproduction / developmental toxicity screening test with 2-EHTG in the presence of marked general toxicity (mortality).

 

Also, the increase infetal variations observed in the dermal prenatal developmental toxicity test with NaTG (slightly increased incidences of extra or rudimentary ribs on Lumbar I and of dumbbell cartilage and bipartite ossification in thoracic centra - common skeletal variations in term rat fetuses), is interpreted as secondary to intrauterine growth retardation resulting from maternal toxicity, especially in combination with the absence of teratogenic alterations and with reductions in maternal and fetal weight.

 

Similar effects were seen for the members of the 3-MPA family. Lower mean male fetal weight, increased incidence of fetuses showing incomplete ossification of the thoracic centrum or less than four ossified caudal vertebrae, increased incidence of absent renal papilla were observed in the prenatal developmental toxicity study with PETMP in the high dose in the presence of marked general toxicity in dams.

 

E12 caused no developmental toxicity in rat, mouse, hamster, and rabbit.

 

Overall, it can be stated, that this whole group of substances has a very low potential to cause reproductive or developmental toxicity. Effects (e.g., lower fetal weight, intrauterine growth retardation, reduced postnatal survival) were observed only in the presence of severe general maternal toxicity.

 

Based on the available data, this group of substances does not need to be classified for developmental toxicity.

 

For better comparison, the NO(A)ELs have been recalculated on the basis of S-content, which is assumed to be the main driver for toxicity:

 

Overall comparison of NO(A)ELs(developmental toxicity) to S-content.

Family	Substance (study)	NO(A)EL [mg/kg bw/d]	S in molecule [%]	Related to S-content [mg/kg bw/d]
ATG	ATG (NOEL, OECD TG 414, oral: gavage, rat)	75	32.1	24.1
TGA	NaTG (NOAEL, OECD TG 416, oral: gavage, rat)	20	30.7	6.1
	GMT (reproduction NOEL, OECD TG 422, oral, rat)	50	21.1	10.5
	2-EHTG (reproduction NOAEL, OECD TG 421, oral: gavage, general toxicity, rat)	50	17.1	8.6
3-MPA	MMP (reproduction NOAEL, OECD TG 422, oral: gavage, rat)	100	29.1	29.2
	PETMP (developmental NOAEL, OECD TG 414, oral: gavage, rat)	120	7.2	8.6
Intra-molecular-S	E12 (developmental NOAEL, OECD TG 414, oral: gavage, rat, mouse, hamster, rabbit)	>/=1600	6.8	108.8

 

After adjustment to S-content, the NOAELs of most of the substances were within the same order of magnitude. Using the lowest NOAEL obtained in the 2-generation reproduction toxicity study conducted with NaTG is considered to be an appropriate starting point for DNEL derivation. Remaining uncertainties due to read-across can be adequately considered by applying an additional assessment factor.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available key studies are reliable or reliable with restrictions (Klimisch 1 – 2) and were conducted according to or similar to guidelines.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

For the assessment of prenatal developmental toxicity, data are available from ATG, NaTG, GMT, 2-EHTG, MMP, PETMP, TLA, and E12. A justification for read-across is attached to IUCLID section 13.

 

In the dose range finding study for the prenatal developmental toxicity test with ATG mortality was observed at 100 mg/kg bw/d and above (3/5 females at 100 mg/kg bw/d, 5/5 females at 150 mg/kg bw/d died). At 100 mg/kg bw/d, the surviving animals had a lower terminal body weight. Increased fetal loss and a lower fetal body weight were observed at 100 mg/kg bw/d, both considered secondary to maternal toxicity.

Based on these results, the dose levels for the prenatal developmental toxicity study with ATG were selected (3, 15, 75 mg/kg bw/d). At the high dose, 2/25 animals died; a cause of death could, however, not be established. All animals of the high dose showed rooting in the bedding material for about 15 min after dosing.

No effects on body weight development, food or water consumption were observed in any dose group. A slightly, but not significantly, lower uterus weight in the high dose females was considered to be incidental. Treatment with ATG had no effects on the numbers of corpora lutea, implantations, live fetuses, dead fetuses, or complete/early/late resorptions. Fetal weights and sex distribution were comparable to control. There was no increase in the frequency of malformations in any dose group. NOEL(embryo-fetal toxicity) = 75 mg/kg bw/d; NOEL(maternal toxicity) = 15 mg/kg bw/d.

 

In the reproduction/developmental toxicity screening test with NaTG (0, 20, 40, 80 mg/kg bw/d) mortality was observed in the high dose (2 males + 6 females; one further female sacrificed due to dead pups) and mid dose (1 female). There were no treatment related effects estrous cyclicity, time to mating, pregnancy index. Females given 80 mg/kg bw/d had a significantly longer gestation period (22.8, p<0.01,vs.21.6 days), a non-significantly lower number of corpora lutea (mean of 6.7, ns,vs.8.5) and a significantly lower number of implantations (10.3, p<0.001,vs.16.5) and pups (9.0, p<0.01,vs.14.7). One female had total resorptions and one litter died on day 1post-partum. There were no treatment-related pup clinical signs or necropsy findings. Pups treated at 40 or 80 mg/kg bw/d had higher mean body weight gains than the controls between day 1 and day 5 post-partum. There were no effects of treatment on sperm morphology, motility or counts. The mean absolute seminal vesicle weights were statistically significantly lower for all male groups in a dose-related manner (absolute weights were -17%, -19% and -35% at 20, 40 and 80 mg/kg/day, respectively). This correlated with a slight decrease in secretory content in the seminal vesicles observed at microscopic examination of the males given 80 mg/kg/day. In the absence of atrophy of seminal epithelium at microscopic examination, these minor findings were not considered to be adverse.

Male reproductive performance was not affected by treatment with NaTG. Dosing at 40 and 80 mg/kg bw/d resulted in deaths in late gestation associated with delayed delivery. NOEL(female reproductive performance) = 20 mg/kg bw/d, NOEL(toxic effects on progeny) = 40 mg/kg bw/d

 

In the 2-generation reproduction toxicity test with NaTG (0, 10, 20, 40 mg/kg bw/d) 40 mg/kg bw/d had no effect on non-pregnant, naïve, adult rats but that it causes maternal toxicity and death of susceptible pregnant females around the time of delivery.

Effects on the dam include lack of nesting/nursing behavior and this causes death of

the pups which have been delivered. If treatment is stopped just prior to delivery, the females may survive delivery but pup death may still occur and pup clinical signs of coldness suggest that maternal nesting/nursing behavior is still impaired by treatment with the test item and affect pup survival. There is evidence that female rats are more affected by test item treatment than males as shown by lower F1 female body weight and body weight gain.

Minimal to moderate periportal heptocellular microvacuolation was observed in females and some male F0 animals treated at 40 mg/kg bw/d suggestive of mild hepatotoxicity. There were no effects on sperm parameters in the control or high-dose group males of either generation. There were no effects of treatment on any parameters measured in either males or females with the test item at 10 or 20 mg/kg bw/d.

The small effects observed on pup survival at 40 mg/kg bw/d may have been secondary to the severe and lethal effects observed in the pregnant dams at that dose-level.

NOEL(parental toxicity, female fertility and gestation, development, growth and survival of the progeny) = 20 mg/kg bw/d

NOEL(male fertility, female mating behavior) >/= 40 mg/kg bw/d

 

Two prenatal developmental toxicity studies with dermal administration of NaTG are available, one in rat (0, 50, 100, 200 mg/kg bw/d) and one in rabbit (0, 10, 15, 25, 65 mg/kg bw/d). Feed consumption was increased in the absence of increased maternal body weights at 50 and 100 mg/kg/day, and in the presence of reduced maternal body weights at 200 mg/kg/day, in rats. There was no effect of treatment in rabbits in maternal body weight, weight changes during gestation, or feed consumption. There was a clear increase in incidence and severity of both erythema and edema at the site of application in all treated groups. Embryo/fetal development was not adversely effected in either species, except for reduced fetal body weight at the high dose in the rat study. In rats, there was also a significant dose-related upward trend for percent fetuses with skeletal variations per litter, and for percent males, but not females, with variations per litter. This was due to slightly increased incidences of extra or rudimentary ribs on Lumbar I and of dumbbell cartilage and bipartite ossification in thoracic centra (both very common skeletal variations in term rat fetuses). These fetal variations, in combination with the absence of teratogenic alterations and with reductions in maternal and fetal weight, are interpreted as secondary to intrauterine growth retardation resulting from maternal toxicity (Tyl, 2003).

 

In the combined repeated dose toxicity study with the reproduction/developmental toxicity screening testwith GMT (0, 15, 50, 150 mg/kg bw/d),5 females at 150 mg/kg bw/d died, the cause for death not being evident from histopathology. No effects were observed on mating, offspring litter size and viability, development, clinical signs of the offspring. The NOEL for reproductive effects in males was 150 mg/kg bw/d as assessed by the absence of adverse effects on fertility or on testicular weight and histopathology. Due to the high maternal mortality and toxicity at 150 mg/kg bw/d in the females, which precluded reproductive assessment at that dose level, the NOEL for reproductive toxicity in females is 50 mg/kg bw/d.

 

In the reproduction/developmental toxicity screening test with 2-EHTG (0, 10, 50, 150 mg/kg bw/d) 3 males and 3 females died in the high dose group. No test item-related effects on male and female mating and fertility indices, male copulation index or female conception index, the mean number of days between pairing and coitus, mean gestation lengths, mean numbers of corpora lutea, implantation sites were observed. Mean live litter size in the high dose was reduced in a test item-related manner. Postnatal survival in this group was reduced on PND 0-1 and birth to PND 4, due in part to 1 female with total litter loss on PND 1. Mean male and female pup body weights and body weight gains in the 150 mg/kg/day group were reduced. Several of these pups were also noted as being small. Mean live litter size and postnatal survival in the low and mid dose were unaffected by test item administration. There were no test article-related effects on the general physical condition of the pups or pup body weights in the 10 and 50 mg/kg/day groups. At the necropsy of pups that were found dead, there were no internal findings that were related to parental treatment with the test item. NOAEL(reproductive, developmental, systemic, neonatal toxicity) = 50 mg/kg bw/d

 

In the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test with MMP (0, 25, 50, 100 mg/kg bw/d) no treatment-related effects on precoital time, fertility indices, mean duration of gestation, number of implantations, postimplantation loss, pup survival or litter size from birth through to scheduled sacrifice on day 4 post partum, mean number of corpora lutea per dam were observed. No abnormal findings were noted for pups at first litter check or during the first 4 days post partum. No effects were noted on sex ratios, mean pup weights on day 0 and day 1 post partum, mean pup weight development during the first 4 days post partum. The NOEL for reproduction/developmental toxicity was considered to be 100 mg/kg bw/d.

 

In the prenatal developmental toxicity study with PETMP (0, 50, 120, 300/200 mg/kg bw/d), the high dose dams showed clinical signs (occasional body tremors, clonic convulsions, hunched posture, ataxia, lethargy, pilo-erection, splayed gait and, labored respiration and decreased respiratory rate), reduced body weight, reduced body weight gain, reduced food consumption and slightly lower gravid uterus weight (though not statistically significant). The maternal NOEL is 120 mg/kg bw/day. 

Lower mean male fetal weight, increased incidence of fetuses showing incomplete ossification of the thoracic centrum or less than four ossified caudal vertebrae, increased incidence of absent renal papilla were observed in the high dose in the presence of marked general toxicity in dams. The developmental NOEL is 120 mg/kg bw/day.

 

In prenatal developmental toxicity studies in rat, mouse, hamster, and rabbit, no adverse effects with respect to number of implantations and maternal or fetal death were noted after oral administration of E12 up to the highest dose level (1600 mg/kg bw/d in rat, mouse, and hamster; 1000 mg/kg bw/d in rabbit). There were no significant differences in numbers of abnormalities of the soft or skeletal tissues between the treated and control fetuses.

 

Summary

With the exception of the mercaptan family, data on prenatal developmental toxicity are available for all subgroups.

 

None of the substances showed developmental toxicity or teratogenicity.

 

The effects observed in thereproduction/developmental toxicity screening test and the 2-generation reproduction toxicity test with NaTG consisting of a significantly longer gestation period, a non-significantly lower number of corpora lutea, a significantly lower number of implantations and reduced pup survival were only seen in the presence on severe maternal toxicity including mortality and thus, secondary effects.

In a comparable manner, mean live litter size, postnatal survival and pup body weights and body weight gains were reduced in the high dose groups of thereproduction / developmental toxicity screening test with 2-EHTG in the presence of marked general toxicity (mortality).

 

Also, the increase infetal variations observed in the dermal prenatal developmental toxicity test with NaTG (slightly increased incidences of extra or rudimentary ribs on Lumbar I and of dumbbell cartilage and bipartite ossification in thoracic centra - common skeletal variations in term rat fetuses), is interpreted as secondary to intrauterine growth retardation resulting from maternal toxicity, especially in combination with the absence of teratogenic alterations and with reductions in maternal and fetal weight.

 

Similar effects were seen for the members of the 3-MPA family. Lower mean male fetal weight, increased incidence of fetuses showing incomplete ossification of the thoracic centrum or less than four ossified caudal vertebrae, increased incidence of absent renal papilla were observed in the prenatal developmental toxicity study with PETMP in the high dose in the presence of marked general toxicity in dams.

 

E12 caused no developmental toxicity in rat, mouse, hamster, and rabbit.

 

Overall, it can be stated, that this whole group of substances as described inTable1(IUCLID chapter 13)has a very low potential to cause reproductive or developmental toxicity. Effects (e.g., lower fetal weight,intrauterine growth retardation,reduced postnatal survival) were observed only in the presence of severe general maternal toxicity.

 

Based on the available data, this group of substances does not need to be classified for developmental toxicity.

 

For better comparison, the NO(A)ELs have been recalculated on the basis of S-content, which is assumed to be the main driver for toxicity:

 

Overall comparison of NO(A)ELs(developmental toxicity) to S-content.

Family	Substance (study)	NO(A)EL [mg/kg bw/d]	S in molecule [%]	Related to S-content [mg/kg bw/d]
ATG	ATG (NOEL, OECD TG 414, oral: gavage, rat)	75	32.1	24.1
TGA	NaTG (NOAEL, OECD TG 416, oral: gavage, rat)	20	30.7	6.1
	GMT (reproduction NOEL, OECD TG 422, oral, rat)	50	21.1	10.5
	2-EHTG (reproduction NOAEL, OECD TG 421, oral: gavage, general toxicity, rat)	50	17.1	8.6
3-MPA	MMP (reproduction NOAEL, OECD TG 422, oral: gavage, rat)	100	29.1	29.2
	PETMP (developmental NOAEL, OECD TG 414, oral: gavage, rat)	120	7.2	8.6
Intra-molecular-Sulfur	E12 (developmental NOAEL, OECD TG 414, oral: gavage, rat, mouse, hamster, rabbit)	>/=1600	6.8	108.8

 

After adjustment to S-content, the NOAELs of most of the substances were within the same order of magnitude. Using the lowest NOAEL obtained in the 2-generation reproduction toxicity study conducted with NaTG is considered to be an appropriate starting point for DNEL derivation. Remaining uncertainties due to read-across can be adequately considered by applying an additional assessment factor.

 

References

Arkema, 2010. Two-generation reproduction toxicity study by oral route (gavage) in rats. Unpublished report. CIT 35047 RSR

 

Bruno Bock, 2010b. Reproduction/developmental toxicity screening test by oral route (gavage) in rats. Unpublished report. CIT 30721 RSR

 

Bruno Bock, 2015c. PETMP: Oral (Gavage) Pre-Natal Development Toxicity Study in the Rat.Unpublished report. Harlan41401135

 

Merck, 1998a. Toxicology – Study report – Reproductive toxicity. Ammonium thioglycolate - dose finding embryo-fetal toxicity study with oral administration to rats. Unpublished report. Merck T9412

 

Merck, 1998b. Toxicology – Study report – Reproductive toxicity. Embryo-fetal toxicity study with oral administration of Ammonium thioglycolate to rats. Unpublished report. Merck T9413

 

Thioesters Association, 2001a. IUCLID data set for 3,3’-thiodipropionic acid, didodecyl ester, submission to US EPA

 

Thioesters Association, 2001b. IUCLID data set for 3,3’-thiodipropionic acid, dioctadecyl ester, submission to US EPA

 

Thioesters Association, 2005. 2-Ethylhexyl mercaptoacetate [CAS No. 7659-86-1]: a reproduction/developmental toxicity screening study in rats. Unpublished report. WIL-528002

 

Thioesters Association, 2007a. Glyceryl thioglycolate 80%: oral (gavage) combined repeat dose toxicity study with reproduction/developmental toxicity screening test in the rat. Unpublished report. SPL 2207/0003

 

Thioesters Association, 2007b. Methyl 3-Mercaptopropionate: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat. Unpublished report. RCC A74698

 

Tyl RW et al., 2003.Developmental Toxicity Evaluation of Sodium Thioglycolate Administered Topically to Sprague–Dawley (CD) Rats and New Zealand White Rabbits. Birth Defects Research (Part B) 68:144–161 (2003)

Justification for classification or non-classification

Overall, mercaptoacetic acid and its salts and esters are not considered to be developmental or reproductive toxicants, except at dose levels associated with maternal lethality.

Maternal mortality was observed in the OECD 421 and OECD 416 studies in rats at doses of 40 and 80 mg/kg bw/day. The acute oral LD50 of sodium mercaptoacetate (NaTG) is 50 – 200 mg/kg bw in fasted animals and the lethal doses in the reproductive toxicity studies fall into this range.

Thus, the mortalities of pregnant dams cannot be regarded as reproductive toxicity, but rather a direct consequence of the established toxic mode of action of mercaptoacetates. Mercaptoacetates are known inhibitors of mitochondrial fatty acid β-oxidation. Studies with NaTG have shown that hypoglycaemia occurs in rats dosed with NaTG via oral gavage and that this effect is pronounced in fasted compared to non-fasted animals (Grosdidier 2011, Report No. 37043, IUCLID Section 7.9.4). The hypoglycaemia is a result of impaired gluconeogenesis secondary to inhibition of fatty acid catabolism by mercaptoacetate. Ad-libitum feeding, as employed in the OECD 421 and OECD 416 studies, apparently saves the animals from succumbing already after a single gavage dose by providing a sufficient external glucose supply. Maternal death in NaTG treated dams occurred in late pregnancy (GD 21-23) or early after parturition (PND 1-2). It is likely that the enhanced energy demand due to parturition and / or lactation further exhausts the glucose reserve of the dams ultimately contributing to their death.

A classification as toxic to reproduction is not justified since all maternal and foetal/offspring effects are directly related to the observations leading to a classification as Acute Tox 3 – H301: Toxic if swallowed.